Virulence Plasmid Diversity inClostridium perfringensType D Isolates

Abstract
Clostridium perfringenstype D isolates are important in biodefense and also cause natural enterotoxemias in sheep, goats, and occasionally cattle. In these isolates, the gene (etx) encoding ε-toxin is thought to reside on poorly characterized large plasmids. Type D isolates sometimes also produce other potentially plasmid-encoded toxins, includingC. perfringensenterotoxin and beta2 toxin, encoded by thecpeandcbp2genes, respectively. In the current study we demonstrated that theetx,cpe, andcpb2genes are carried on plasmids in type D isolates and characterized the toxin-encoding plasmids to obtain insight into their genetic organization, potential transferability, and diversity. Southern blotting of pulsed-field gels showed that theetxgene of type D isolates can be present on at least five different plasmids, whose sizes range from 48 to 110 kb. Theetxplasmids also typically carried IS1151andtcpopen reading frames (ORFs) known to mediate conjugative transfer ofC. perfringensplasmid pCW3. PCR studies revealed that other than theirtcpORFs,etxplasmids of type D isolates do not carry substantial portions of the conserved or variable regions in thecpeplasmids of type A isolates. Southern blotting also demonstrated that in type D isolates thecpeandcpb2genes are sometimes present on theetxplasmid. Collectively, these findings confirmed that the virulence of type D isolates is heavily plasmid dependent and indicated that (i) a single type D isolate can carry multiple virulence plasmids, (ii) a single type D virulence plasmid can carry up to three different toxin genes, and (iii) manyetxplasmids should be capable of conjugative transfer.